Anti pd-l1 antibody and use thereof

ABSTRACT

Provided in the present invention is a PD-L1 immunosuppressant, and specifically provided are a monoclonal antibody capable of targeting PD-L1, and nucleotides, combinations of polynucleotides, expression vectors and combinations of expression vectors encoding the antibody. Also provided in the present invention is a conjugate or pharmaceutical composition containing the above-mentioned anti PD-L1 antibody. Further provided in the present invention is the use of the above-mentioned anti PD-L1 antibody, nucleotides, combinations of polynucleotides, expression vectors, combinations of expression vectors, conjugate or pharmaceutical composition in the preparation of a medicament for treating or preventing cancers.

FIELD

The present invention relates to the field of biomedicine, specificallyto an anti-PD-L1 antibody or antigen-binding fragment thereof, andmedical use thereof.

BACKGROUND

Recently, in the field of tumor therapy, increasing efforts have beendedicated to utilizing the body's immune system to defense tumors. Thismethod of inhibition and killing of tumor cells by mobilizing the body'simmune system is called as tumor immunotherapy. Tumor immunotherapy,including cell immunotherapy, tumor vaccines, passive immunotherapytargeting tumors, and immune checkpoint inhibitors, is currently themost promising research direction in the field of tumor therapy and hasyielded a number of prospective research results.

Immune checkpoint refers to a signaling pathway that controls theintensity of T cell immune response by balancing costimulatory andco-suppressive signals (Reference 1). Immune checkpoints can maintainimmune tolerance by regulating the intensity of the autoimmune responseunder normal circumstances. When the body is invaded by tumors, however,the activation of immune checkpoints can inhibit autoimmunity, whichfavors the growth and escape of tumor cells. Immune checkpoints such asCTLA-4, programmed death receptor-1 (PD-1)/programmed death ligand-1(PD-L1) and TLM-3 are the key negatively regulatory molecules, playingan important role in tumors' immune evasion. Blocking the negativelyregulatory pathway of immune checkpoints with a specific antibody andrebuilding the ability of body's immune system to recognize and killtumor cells have achieved good therapeutic outcomes in tumorimmunotherapy. Among the known immune checkpoints, PD-1/PD-L1 has drawnhigh attention in tumor immune research and therapy.

PD-1 (programmed death 1, programmed death receptor 1, CD279), a memberof CD28 superfamily, is an important immunosuppressive molecule. PD-1 isexpressed on activated T cells, B cells, NK cells, monocytes and sometumor cells. PD-1 is a transmembrane protein of 288 amino acids encodedby PDCD1 gene. PD-1 mainly comprises an extracellularregion—immunoglobulin variable region (IgV)-like domain, a transmembraneregion and an intracellular region. The intracellular region comprisesC-terminal and N-terminal amino acid residues, and contains two separatephosphorylation sites located in immunoreceptor tyrosine basedinhibitory motif (ITIM) and immunoreceptor tyrosine based switch motif(ITSM). The binding of the extracellular IgV-like domain of PD-1 to itsligand allows changes in ITSM followed by recruitment of SHP2 signals,resulting in the activation of downstream pathways (Reference 2).

There are two natural ligands for PD-1, PD-L1 and PD-L2 (Reference 3).PD-L1 (Programmed death ligand 1, CD274, B7-H1) is a 40 kDatransmembrane protein encoded by CD274 gene, and induced to be expressedon T cells, B cells, dendritic cells, macrophages, mesenchymal stemcells, bone marrow-derived mast cells and non-hematopoietic cells, andit may be rapidly upregulated in tumor tissues and other tissues inresponse to interferon and other inflammatory factors (Reference 4).Upon activation of PD-1/PD-L1 pathway, immune system is suppressed incancer, pregnancy, tissue transplantation and autoimmune diseases. PD-L2(Programmed death ligand 2, CD273, B7-DC) is limited to be expressed andupregulated mainly in activated macrophages, dendritic cells, and mastcells (Reference 5). Although PD-L1 and PD-L2 share 37% sequencehomology, their regulatory effects are different due to the differencein their main expression cells. Unlike PD-L2, PD-L1 is expressed on avariety of tumor cells, making PD-L1 being a main ligand for studyingPD-1/PD-L pathway in the field of tumor immunotherapy.

Since PD-L1 can also bind to CD80 (belonging to the immunoglobulinsuperfamily, CD28 and CTLA4 as its ligands, playing an important role inautoimmune monitoring, humoral immune response and transplantationresponse) (Reference 6), inhibition of PD-L1 may relieve theinterference with CD80 to thereby enhance T cell activity. From theperspective of drug safety, PD-L2, another receptor of PD-1, has anaffinity for PD-1 that is three times higher than the affinity of PD-L1for PD-1 (Reference 7), and PD-L1 blockers do not bind to PD-L2.Theoretically, a PD-1 antibody blocks the interaction of PD-1 with bothPD-L1 and PD-L2, while a PD-L1 antibody only blocks the interaction ofPD-1 with PD-L1, reserving the interaction of PD-1 with PD-L2. And also,PD-L2 is essential for maintaining the immune tolerance in the lung andgastrointestinal tract. Taken together, a PD-L1 antibody may have fewerside effects on lung and gastrointestinal tract than a PD-1 antibody. Ascompared with PD-1 blockers, PD-L1 blockers may have a betterperformance in terms of effectiveness and safety (Reference 8).

Currently, three PD-L1 antibody drugs have been approved by US FDA formarketing (as shown in Table 1).

TABLE 1 PD-L1 immune checkpoint inhibitors approved by FDA Time to Mainapproved Trade name Drug market Manufacturer indications TecentrigAtezolizumab 2016 Roche Urothelial cancer, Bavencio Avelumab 2017Merck/Pfizer non-small cell lung Imfinzi Durvalumab 2017 AstraZenecacancer and Merkel cell carcinoma

Atezolizumab was approved by US FDA for the first time in May 2016, andwas approved for many indications in the following two years (as shownin Table 2).

TABLE 2 Approved indications of Atezolizumab Time Approved indications2016.05.18 (1) Patients who develop disease progression during or afterplatinum-containing chemotherapy, and (2) Patients with locally advancedor metastatic urothelial cancer who develop disease progression within12 months after platinum-containing chemotherapy as a neoadjuvant oradjuvant treatment 2016.10.18 Patients with metastatic non-small celllung cancer (NSCLC) who develop progression after platinum-containingchemotherapy 2017.04.17 (1) Patients not suitable forcisplatin-containing chemotherapy, and (2) Patients with locallyadvanced or metastatic urothelial cancer who develop disease progressionduring or after any platinum-containing chemotherapy, or within 12months after a neoadjuvant or adjuvant chemotherapy 2018.06.19 (1)Patients not suitable for cisplatin-containing chemotherapy and withtumors expressing PD-L1 (PD-L1-stained tumor infiltrating immunocyte(IC) covering ≥5% of the tumor area), (2) Patients with tumorsexpressing PD-L1 but not meeting any platinum-containing chemotherapyconditions, and (3) Patients with locally advanced or metastaticurothelial cancer who develop disease progression during or after anyplatinum- containing chemotherapy, or within 12 months after aneoadjuvant or adjuvant chemotherapy 2018.07.02 (1) Patients notsuitable for cisplatin-containing chemotherapy by FDA-approved test andwith tumors expressing PD-L1 (PD-L1-stained tumor infiltratingimmunocyte (IC) covering ≥5% of the tumor area), (2) Patients notmeeting any platinum- containing chemotherapy conditions, and (3)Patients with locally advanced or metastatic urothelial cancer whodevelop disease progression during or after any platinum-containingchemotherapy, or within 12 months after a neoadjuvant or adjuvantchemotherapy

In 2019, Atezolizumab was further approved for the first-line treatmentof three refractory advanced cancers, includingAtezolizumab+Bevacizumab+chemotherapy approved by European Union for thefirst-line treatment of advanced non-squamous non-small cell lungcancer, and a combination of Atezolizumab with chemotherapy approved bythe United States for the first-line treatment of PD-L1-positiveadvanced triple-negative breast cancer as well as for the first-linetreatment of advanced small cell lung cancer.

The other two PD-L1 antibody drugs on the market, Avelumab andDurvalumab, have also been approved successively for the treatment offor example metastatic MERKEL cell carcinoma, locally advanced ormetastatic urothelial cancer, or surgically unresectable stage IIInon-small cell lung cancer. See Tables 3-4 for details.

TABLE 3 Approved indications of Avelumab Time Approved indications2017.03.23 Patients with metastatic MERKEL cell carcinoma (MCC)2017.05.09 Patients with locally advanced or metastatic urothelialcancer (mUC) who develop disease progression during or afterplatinum-containing chemotherapy 2017.05.09 Patients with locallyadvanced or metastatic urothelial cancer (mUC) who develop diseaseprogression within 12 months after platinum-containing chemotherapybefore surgery (neoadjuvant treatment) or after surgery (adjuvanttreatment)

TABLE 4 Approved indications of Durvalumab Time Approved indications2017.05.01 Patients with locally advanced or metastatic urothelialcancer who develop disease progression 12 months after platinum-containing chemotherapy or adjuvant chemotherapy 2018.02.16 Patientswith surgically unresectable stage III non-small cell lung cancer, andwith no amelioration of disease in the concurrent treatment withplatinum-containing chemotherapy and radiotherapy

Patent application CN102245640A also disclosed a PD-L1 antibody and usethereof for enhancing T cell function to upregulate cell-mediated immuneresponse and provided a method for the treatment of T cell dysfunction,including infection (e.g. acute and chronic) and tumor immunization.Cancers targeted by tumor immunization include breast cancer, lungcancer, colon cancer, ovarian cancer, melanoma, bladder cancer, kidneycancer, liver cancer, salivary cancer, stomach cancer, glioma, thyroidcancer, thymic cancer, epithelial cancer, head and neck cancer, gastricand pancreatic cancer.

From the current approved indications of each PD-L1 antibody and theexisting technical literatures, different PD-L1 antibodies are directedto totally different indications, wherein three marketed antibodies haveexperienced several failures in phase III clinical trial, such asBavencio's three trials for indications of ovarian cancer: JAVELINOvarian 100, JAVELIN Ovarian 200 and JAVELIN Ovarian PARP 100,suggesting there are still a large number of substantial clinical needsthat have not been met currently. Therefore, there is clinical urgencyin developing more PD L1 inhibitors that are more effective andapplicable to more indications, especially monoclonal antibodiestargeting PD-L1.

SUMMARY

The present invention provides an anti-PD-L1 antibody, as well as apolynucleotide, a polynucleotide combination, an expression vector, andan expression vector combination encoding the antibody. The presentinvention further provides a conjugate or a pharmaceutical compositioncomprising the above-mentioned anti-PD-L1 antibody. The presentinvention further provides use of the above-mentioned polynucleotide,polynucleotide combination, expression vector, expression vectorcombination, conjugate or pharmaceutical composition of the anti-PD-L1antibody for a medicament for treatment or prevention of cancer.

The present invention provides an isolated anti-PD-L1 antibody orantigen-binding fragment thereof, wherein the anti-PD-L1 antibody orantigen-binding fragment thereof comprises a heavy chain variable regionand a light chain variable region. The heavy chain variable regionand/or the light chain variable region comprises a CDR sequenceidentical to that of an antibody defined by the following sequence orobtained by 1-2 amino acid substitutions of the CDR sequence of theantibody defined by the following sequence:

(1) an amino acid sequence of a heavy chain variable region as shown inSEQ ID NO: 31; and/or(2) an amino acid sequence of a light chain variable region as shown inSEQ ID NO: 32.

In a specific embodiment, according to different determination methodsor system identifications, the complementarity determining regions CDRs1-3 of the corresponding heavy chain and light chain variable regionsare as shown in the Table 5.

TABLE 5 CDRs 1-3 Amino acid sequences of heavy chain andlight chain variable regions Category System CDR1 CDR2 CDR3 Heavy IMGTSEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 chain GFSLSRYS IWGVGTTARNWGTADYFDY Kabat SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 RYSVHMIWGVGTTDYNSALKS NWGTADYFDY Chothia SEQ ID NO: 13 SEQ ID NO: 14SEQ ID NO: 15 GFSLSRY WGVGT NWGTADYFDY AbM SEQ ID NO: 19 SEQ ID NO: 20SEQ ID NO: 21 GFSLSRYSVH MIWGVGTTD NWGTADYFDY Contact SEQ ID NO: 25SEQ ID NO: 26 SEQ ID NO: 27 SRYSVH WLGMIWGVGTTD ARNWGTADYFD Light IMGTSEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 chain KSVHTSGYSY LAS QHSGELPYTKabat SEQ ID NO: 10 SEQ ID NO: 11 SEQ ID NO: 12 RASKSVHTSGYSYMH LASNLESQHSGELPYT Chothia SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18RASKSVHTSGYSYMH LASNLES QHSGELPYT AbM SEQ ID NO: 22 SEQ ID NO: 23SEQ ID NO: 24 RASKSVHTSGYSYMH LASNLES QHSGELPYT Contact SEQ ID NO: 28SEQ ID NO: 29 SEQ ID NO: 30 HTSGYSYMHWY LLIYLASNLE QHSGELPY

Further, the present invention provides an isolated anti-PD-L1 antibodyor antigen-binding fragment thereof, which in some specific embodiments,comprises a heavy chain and light chain variable region, wherein:

(1) for the heavy chain variable region, CDR1 comprises an amino acidsequence as shown in SEQ ID NO: 1, 7, 13, 19 or 25 or obtained by 1 or 2amino acid substitutions of SEQ ID NO: 1, 7, 13, 19 or 25; CDR2comprises an amino acid sequence as shown in SEQ ID NO: 2, 8, 14, 20 or26 or obtained by 1 or 2 amino acid substitutions of SEQ ID NO: 2, 8,14, 20 or 26; CDR3 comprises an amino acid sequence as shown in SEQ IDNO: 3, 9, 15, 21 or 27 or obtained by 1 or 2 amino acid substitutions ofSEQ ID NO: 3, 9, 15, 21 or 27; and/or(2) for the light chain variable region, CDR1 comprises an amino acidsequence as shown in SEQ ID NO: 4, 10, 16, 22 or 28 or obtained by 1 or2 amino acid substitutions of SEQ ID NO: 4, 10, 16, 22 or 28; CDR2comprises an amino acid sequence as shown in SEQ ID NO: 5, 11, 17, 23 or29 or obtained by 1 or 2 amino acid substitutions of SEQ ID NO: 5, 11,17, 23 or 29; CDR3 comprises an amino acid sequence as shown in SEQ IDNO: 6, 12, 18, 24 or 30 or obtained by 1 or 2 amino acid substitutionsof SEQ ID NO: 6, 12, 18, 24 or 30.

Further, the present invention provides an isolated anti-PD-L1 antibodyor antigen-binding fragment thereof. In some specific embodiments,

(1) CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 1-3 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 1-3, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 4-6 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 4-6; or(2) CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 7-9 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 7-9, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 10-12 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 10-12; or(3) CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 13-15 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 13-15, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 16-18 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 16-18; or(4) CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 19-21 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 19-21, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 22-24 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 22-24; or(5) CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 25-27 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 25-27, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 28-30 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 28-30.

In some specific embodiments, the present invention provides an antibodyor antigen-binding fragment thereof, wherein the CDRs 1-3 of the heavychain variable region comprise amino acid sequences of SEQ NOs: 1-3; theCDRs 1-3 of the light chain variable region comprise amino acidsequences of SEQ ID NOs: 4-6.

In some specific embodiments, the present invention provides an antibodyor antigen-binding fragment thereof, comprising variable regionsselected from the following group:

(1) a heavy chain variable region comprising a sequence as shown in SEQID NO: 31, or comprising the same CDRs 1-3 as in SEQ ID NO: 31 and moretha 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:31; and/or(2) a light chain variable region comprising a sequence as shown in SEQID NO: 32, or comprising the same CDRs 1-3 as in SEQ ID NO: 32 and morethan 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:32.

The heavy chain variable region of the anti-PD-L1 antibody of thepresent invention comprises an amino acid sequence of (SEQ ID NO: 31):

QVQLQESGPG LVKPSETLSL TCTVSGESLS RYSVHWIRQP PGKGLEWLGM IWGVGITDYN 60SALKSRLTIS KDTSKNQFSL KLSSVTAADT AVYYCARNWG TADYFDYWGQ GTIVTVSSAS 120

The light chain variable region of the anti-PD-L1 antibody of thepresent invention comprises an amino acid sequence of (SEQ ID NO: 32):

DIVLTQSPAS LAVSPGQRAT ITCRLSKEVH TSGYSIMHWY QQKPGQPPKI LIYLASNLES 60GVPARFSGSG SGTDFTLTIN PVEANDTANY YCQHSGELPY TFGGGTKVEI KRT 113

In some specific embodiments, the present invention provides an antibodyor antigen-binding fragment thereof, comprising (1) a heavy chainvariable region with an sequence as shown in SEQ ID NO: 31; and/or (2) alight chain variable region with a sequence as shown in SEQ ID NO: 32.

In some specific embodiments, the present invention provides an antibodyor antigen-binding fragment thereof, comprising (1) a heavy chain withan amino acid sequence as shown in SEQ ID NO: 33; and/or (2) a lightchain with an amino acid sequence as shown in SEQ ID NO: 34.

The antibody provided by the present invention may be a monoclonalantibody, chimeric antibody, humanized antibody, bispecific antibody,multispecific antibody, or Fab fragment, F(ab′) fragment, F(ab′)₂fragment, Fv fragment, dAb, Fd, single chain antibody (scFv). Further,the antibody is a humanized monoclonal antibody.

The antibody provided by the present invention further comprises a humanor murine constant region, and the constant region may further beselected from IgG1, IgG2, IgG3, and IgG4. The IgG2 comprises IgG2A andIgG2B.

The present invention further provides an isolated polynucleotideencoding the above-mentioned antibody or antigen-binding fragmentthereof, or a polynucleotide combination encoding a heavy chain or apart thereof and a light chain or a part thereof of the above-mentionedantibody or antigen-binding fragment thereof.

The present invention further provides a nucleic acid construct orvector, comprising the above-mentioned polynucleotide encoding theanti-PD-L1 antibody or antigen-binding fragment thereof.

The present invention further provides a host cell comprising theabove-mentioned nucleic acid construct or vector. The cell may be aprokaryotic cell, eukaryotic cell, yeast cell, mammalian cell, E. colicell or CHO cell, NS0 cell, Sp2/0 cell, BHK cell.

The present invention further provides an antibody-drug conjugate,comprising the anti-PD-L1 antibody or antigen-binding fragment thereofof the present invention and a drug toxin. The drug toxin may beselected from chemicals, toxins, polypeptides, enzymes, isotopes,cytokines, antibodies, or other bioactive substances, or a mixturethereof that can inhibit cell growth or kill cells directly, indirectly,or by activating the body's immune response to thereby treat tumors,preferably interleukins, tumor necrosis factors, chemokines,nanoparticles, MMAE, MMAF, DM1, DM4, calicheamicin, duocarmycin,doxonibicin.

The present invention further provides a pharmaceutical composition,comprising the anti-PD-L1 antibody or antigen-binding fragment thereofand/or the above-mentioned conjugate of the present invention, and apharmaceutically acceptable carrier.

The present invention further provides a method for the manufacture ofan anti-PD-L1 antibody, comprising culturing the above host cell under acondition suitable for a vector encoding the anti-PD-L1 antibody orantigen-binding fragment to be expressed, and recovering the antibody orfragment.

The present invention further provides a method for enhancing T cellfunction, comprising administering an effective amount of theabove-mentioned pharmaceutical composition of the present invention todysfunctional T cells.

In another aspect, the present invention provides a method for thetreatment or prevention of cancer, comprising administering atherapeutically effective amount of the antibody, polynucleotide,polynucleotide combination, expression vector, conjugate and/orpharmaceutical composition of the present invention to a subject in needthereof.

In yet another aspect, the present invention provides use of theanti-PD-L1 antibody or antigen-binding fragment thereof, thepolynucleotide, the polynucleotide combination, the correspondingnucleic acid construct or vector encoding the antibody orantigen-binding fragment thereof, the antibody-drug conjugate or thepharmaceutical composition of the present invention for the manufactureof a medicament for use in the treatment or prevention of cancer.

In yet another aspect, the present invention provides the antibody,polynucleotide, polynucleotide combination, expression vector, conjugateand/or pharmaceutical composition of the present invention, for use inthe treatment or prevention of cancer.

Further, the cancer is a solid tumor.

Further, the solid tumor is lung cancer, colorectal cancer, breastcancer, ovarian cancer, melanoma, bladder cancer, urothelial cancer,kidney cancer, liver cancer, salivary cancer, stomach cancer, gliomas,thyroid cancer, thymic cancer, epithelial cancer, head and neck cancer,gastric and pancreatic cancer.

Further, the lung cancer is non-small cell lung cancer.

Further, the ovarian cancer is triple negative breast cancer.

Yet another aspect of the present invention provides a method for thetreatment of T cell dysfunction, comprising administering atherapeutically effective amount of the above-mentioned pharmaceuticalcomposition of the present invention to a patient with T celldysfunction. The T cell dysfunction comprises infection and tumorimmunization. The tumor immunization is caused by a cancer selected frombreast cancer, lung cancer, colon cancer, ovarian cancer, melanoma,bladder cancer, kidney cancer, liver cancer, salivary cancer, stomachcancer, glioma, thyroid cancer, thymic cancer, epithelial cancer, headand neck cancer, and gastric and pancreatic cancer.

The present invention further provides use of the anti-PD-L1 antibody orantigen-binding fragment thereof, the polynucleotide, the polynucleotidecombination, the corresponding nucleic acid construct or vector encodingthe antibody or antigen-binding fragment thereof, the antibody-drugconjugate or the pharmaceutical composition of the present invention forthe manufacture of a medicament for use in treatment or prevention ofcancer.

The anti-PD-L1 monoclonal antibody provided by the present invention isa novel monoclonal antibody targeting PD-L1 with a new CDR sequence andamino acid sequence. The anti-PD-L1 monoclonal antibody provided by thepresent invention has a surprising affinity and specificity to humanPD-L1, and has significant advantages in competing with human PD-1 tobind to human PD-L1. The anti-PD-L1 monoclonal antibody provided by thepresent invention exhibits unexpected tumor inhibitory effects in invitro animal models of both non-small cell lung cancer and colorectalcancer, and provides a better option for inhibiting tumor progression.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an affinity binding and dissociation curve of hAAG5D8 tohuman PD-L1, where the curve 1 indicates 100 nM hAAG5D8, the curve 2indicates 50 nM hAAG5D8, and the curve 3 indicates 2.5 nM hAAG5D8.

FIG. 2 shows a curve of competing between hAAG5D8 and human PD-1 forbinding.

FIG. 3 shows the binding ability of hAAG5D8 to various B7 familyproteins (PD-L1, PD-L2, B7-H3, PD-1 and CD80).

FIG. 4 shows the growth curve of tumor after administration of hAAG5D8(5 mg/kg), MPDL3820A (5 mg/kg, positive control) or human IgG1 (5 mg/kg,negative control) in a subcutaneous xenograft tumor model of humannon-small cell lung cancer.

FIG. 5 shows the change in tumor volume after administration of hAAG5D8(1 mg/kg, 3 mg/kg, or 9 mg/kg), MPDL3820A (10 mg/kg, positive control)or PBS (negative control) in a subcutaneous xenograft tumor model ofhuman colorectal cancer.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as understood by those ordinarily skilled in theart. With regard to the definitions and terms in the art, reference maybe made to Current Protocols in Molecular Biology (Ausubel). Thestandard three- and/or one-letter code used for expressing one of 20common L-amino acids in the art is adopted as the abbreviation of anamino acid residue.

In the present invention, a method for determining or numbering thecomplementarity determining region (CDR) of an antibody's variabledomain includes IMGT, Kabat, Chothia, AbM and Contact, which are wellknown in the art.

For the purposes of the present invention, the “consistency”, “identity”or “similarity” between two nucleic acid or amino acid sequences refersto the percentage of identical nucleotides or identical amino acidresidues between the two sequences to be compared after optimalalignment. The percentage is purely statistical and the differencesbetween the two sequences are randomly distributed and cover their fulllength. Sequence comparison between two nucleic acid or amino acidsequences are usually performed by comparing these sequences after theyhave been optimally matched, and the comparison can be performed on asegment or on a “comparison window”. In addition to manualimplementation, the optimal alignment for comparing sequences can alsobe performed by the local homology algorithm of Smith and Waterman(1981) [Ad. App. Math. 2: 482], the local homology algorithm ofNeddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], the similaritysearch method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA85: 2444), or a computer software using these algorithms (GAP, BESTFIT,FASTA and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575Science Dr., Madison, Wis., or BLAST N or BLAST Pcomparison software).

As used herein, “antibody” is used in a broadest sense and encompassesvarious antibodies including, but not limited to, a monoclonal antibody,a polyclonal antibody, and a multispecific antibody (e.g., a bispecificantibody). As used herein, “antigen-binding fragment” refers to anantibody fragment consisting of or comprising a partial sequence of aheavy or light variable chain of an antibody from which it is derived,wherein the partial sequence is capable of retaining the same bindingspecificity as the antibody from which it is derived and a sufficientaffinity, preferably equal to at least 1/100, more preferably at least1/10 of the affinity of the antibody from which it is derived. Such afunctional fragment comprises a minimum of 5 amino acids, preferably 10,15, 25, 50 or 100 contiguous amino acids of the antibody sequence fromwhich it is derived, including (particularly) Fab, F(ab′), F(ab′)₂, Fv,dAb, Fd, a complementarity determining region (CDR) fragment, a singlechain antibody (scFv), and a bivalent single chain antibody, thatcontains at least an immunoglobulin fragment enough to allow a specificantigen to bind to the polypeptide. The above fragments can be preparedby a synthetic or enzymatic method, or by chemical cleavage of an intactimmunoglobulin, or can be genetically engineered by recombinant DNAtechnology. The preparation methods thereof are well known in the art. Aheavy chain contains a heavy chain variable region (abbreviated as VH)and a heavy chain constant region. The heavy chain constant regioncontains three domains, CH1, CH2 and CH 3. A light chain contains alight chain variable region (abbreviated as VL) and a light chainconstant region. The light chain constant region contains a domain, CL.VH and VL regions can be further subdivided into multiple regions withhigh variability, called as complementarity determining regions (CDRs),interspersed with more conservative regions called as framework regions(FRs). Each VH and VL is composed of three CDRs and four FRs, which arearranged from the amino terminal to the carboxy terminal in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variableregions of the heavy and light chains contain a binding domain thatinteracts with an antigen. The constant region of an antibody canmediate binding of an immunoglobulin to a host tissue or factor,including various cells in the immune system (such as effector cells)and the first component of the classical complement system (Clq).Chimeric or humanized antibodies are also encompassed by the antibodiesaccording to the present invention.

The term “humanized antibody” refers to an antibody that contains a CDRregion derived from a non-human antibody, with the rest deriving fromone (or several) human antibody. Moreover, in order to retain bindingaffinity, some residues at the backbone (called FR) segment can bemodified (Jones et al., Nature, 321: 522-525, 1986; Verhoeyen et al.,Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332: 323-327,1988). Humanized antibodies or fragments thereof according to thepresent invention can be prepared by techniques known to those skilledin the art (e.g., described in the document Singer et al., J. Immun.150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; or Bebbington et al., Bio/Technology, 10: 169-175, 1992).

The term “chimeric antibody” refers to an antibody in which the variableregion sequence is from one species while the constant region sequenceis from another species, for example, an antibody in which the variableregion sequence is from a mouse antibody while the constant regionsequence is from a human antibody. A chimeric antibody or a fragmentthereof according to the present invention can be prepared by usinggenetic recombination technology. For example, the chimeric antibody canbe produced by cloning a recombinant DNA comprising a promoter and asequence encoding a variable region of a non-human, especially a murinemonoclonal antibody according to the present invention, and a sequenceencoding a constant region of a human antibody. The chimeric antibody ofthe present invention encoded by such a recombinant gene will be, forexample, a murine-human chimera whose specificity is determined by thevariable region derived from murine DNA, and the isotype is determinedby the constant region derived from human DNA. For methods for preparinga chimeric antibody, for example, reference can be made to the documentVerhoeyn et al. (BioEssays, 8:74, 1988).

The term “monoclonal antibody” refers to a preparation of an antibodymolecule consisting of a single molecule. Monoclonal antibodycompositions display a single binding specificity and affinity for aparticular epitope.

The term an “isolated” nucleic acid molecule refers to a nucleic acidmolecule identified and separated from at least one contaminant nucleicacid molecules, and is generally associated with the contaminant nucleicacid molecule in the natural source of an antibody nucleic acid. Anisolated nucleic acid molecule is different in form or environment fromwhen it is found in nature, and therefore different from that existingin natural cells. However, an isolated nucleic acid molecule comprises anucleic acid molecule contained in cells where an antibody is usuallyexpressed, and where for example, it is located on a differentchromosomal position from that in a natural cell.

Generally, in order to prepare a monoclonal antibody or functionalfragment thereof, especially a murine-derived monoclonal antibody orfunctional fragment thereof, reference can be made to the technologyespecially described in the manual “Antibodies” (Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, ColdSpring Harbor N.Y., pp. 726, 1988) or the technique for preparation fromhybridoma cells described by Kohler and Milstein (Nature, 256: 495-497,1975).

EXAMPLES

The embodiments of the present invention will be described in detailbelow in conjunction with examples. However, it will be understood bythose skilled in the art that the following examples are only used toillustrate the present invention and should not be regarded as limitingthe scope of the present invention.

Example 1 Production of Anti-PD-L1 Antibody

Through extensive screening of anti-PD-L1 antibodies obtained afterimmunizing mice, a candidate murine antibody mAAG5D8 was determined.After sequence alignment in the antibody variable region database, ahuman IgG1 framework region with a high homology to the murine PD-L1antibody mAAG5D8 was determined. For mAAG5D8, a variety of humanizedantibodies was further designed and compared for their affinity, andfinally a candidate humanized anti-PD-L1 antibody hAAG5D8 wasdetermined.

TABLE 6 CDRs 1-3 amino acid sequences of heavychain and light chain variable regions of anti-PD-L1 humanized antibodyhAAG5D8 (determined by IMGT method) Heavy chain CDR1 SEQ ID NO: 1GFSLSRYS CDR2 SEQ ID NO: 2 IWGVGTT CDR3 SEQ ID NO: 3 ARNWGTADYFDYLight chain CDR1 SEQ ID NO: 4 KSVHTSGYSY CDR2 SEQ ID NO: 5 LAS CDR3SEQ ID NO: 6 QHSGELPYT

The heavy chain variable region of anti-PD-L1 humanized antibody hAAG5D8comprises an amino acid sequence of (SEQ ID NO: 31):

QVQLQESGPG LVKPSETLSL TCTVSGESLS RYSVHWIROP PGKGLEWLGM IWGVGTTDYN 60SALKSRLTIS KDTSKNQFSL KLSSVTAADT AVYYCARNWG TADYFDTAGQ GTTVTVSSAS 120

The light chain variable region of anti-PD-L1 humanized antibody hAAG5D8comprises an amino acid sequence of (SEQ ID NO: 32):

DIVLTQSPAS LAVSPGQRAT ITCBASKSVH TSGYSYMHWY QQKPGQPPKL LIYLASNLES 60GVPARFSGSG SGTDFTLTIN PVEANDTANY YCQHSGELPY TFGGGTKVEI KRT 113

The amino acid sequences of the heavy chain and light chain ofanti-PD-L1 humanized antibody hAAG5D8 are shown in SEQ ID NO: 33 and SEQID NO: 34, respectively.

Example 2 Comparison of Affinity of hAAG5D8 to Human PD-L1

The affinity of hAAG5D8 to human PD-L1 was detected using biolayerinterferometry (BLI). The wells in A-D columns 1, 3, and 5 of a black96-well plate were added with a PBS solution as baseline 1, baseline 2and a dissociation solution, respectively. The wells in column 2 wereadded with a PD-L1 solution (R&D company), in column 4 with a hAAG5D8solution (The concentrations were as follows: 100 nM, 50 nM, 25 nM, and0 nM), in column 10 with an imidazole solution, in column 11 with water,and in column 12 with a nickel sulfate solution. Ni-NTA probe (Fortebio)was employed in the experiment. First, the probe was dipped in A-Dcolumn 1 for 180 seconds to stabilize the baseline. The Ni-NTA probe wasthen dipped in A-D column 2 for 300 seconds to immobilize PD-L1 on theprobe. The Ni-NTA probe was then dipped in A-D column 3 for 120 secondsto stabilize the baseline. The Ni-NTA probe was then dipped in A-Dcolumn 4 for 600 seconds to allow the binding of hAAG5D8 with adifferent concentration to the immobilized PD-L1 protein on the probe.The Ni-NTA probe was then dipped in A-D column 5 for 600 seconds toallow the antibody to dissociate spontaneously. Finally, the Ni-NTAprobe was dipped in A-D columns 10, 11, and 12 successively to force theimmobilized PD-L1 dissociating from the probe. Data analysis wasperformed by Data analysis 7.0 to obtain the binding and dissociationequilibrium constant K_(D).

Results and conclusions: The binding and dissociation equilibriumconstant K_(D) value and affinity binding and dissociation curve ofhAAG5D8 to human PD-L1 are shown in Table 7 and FIG. 1, respectively.The experimental results show that the affinity of hAAG5D8 to humanPD-L1 is much higher than the affinity of PD-1 to PD-L1 of 8.2 μM(Molecular Interactions of Antibody Drugs Targeting PD-1, PD-L1, andCTLA-4 in Immuno-Oncology, Hyun Tae Lee et al., Molecules 2019, 24,1190; doi:10.3390/molecules24061190, Mar. 26, 2019, last paragraph onpage 4).

TABLE 7 Affinity kinetic constant of anti-PD-L1 antibody binding toPD-L1 Main parameter K_(D)(M) Affinity kinetic constant 7.62E−11 ±3.32E−11

Example 3 Competing Between hAAG5D8 and Human PD-1 for Binding

The ability of hAAG5D8 to compete with PD-1 to bind to PD-L1 was testedby ELISA. PD-L1 (Fc Tag) was diluted to 1 μg/mL with a coating buffer (6mM Na₂CO₃, 14 mM NaHCO₃), and then added 100 μL to each well followed byovernight incubation at 4° C. The plate was then washed with PBST, andadded with 250 μL of a blocking solution (3% BSA/PBST) to each well toblock for 2 h at 25° C. Afterwards, the plate was washed with PBST andadded with 50 μL of human PD-1 at a concentration of 6 μg/mL as well as50 μL of a gradiently diluted hAAG5D8 solution (at a concentration of8000 ng/mL, 2000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL,31.25 ng/mL, 6.25 ng/mL, or 1.25 ng/mL) to each well followed by mixingwell and incubation at 25° C. for 2 h. Next, the plate was washed withPBST and added with 100 μL of an enzyme-labeled secondary antibody (goatanti-human IgG-Fc-HRP diluted 1:5000) to each well, followed byincubation at 25° C. for 1 h. Finally, color development was performedand values were read at 450 nm. A four-parameter equation was used forcurve fitting to calculate the EC₅₀ value.

Results and conclusions: EC₅₀ value and the competitive binding curve ofhAAG5D8 competitively binding to human PD-L1 are shown in Table 8 andFIG. 2, respectively. Experimental results show that hAAG5D8 caneffectively block the binding of PD-1 to PD-L1.

TABLE 8 Competing between hAAG5D8 and human PD-1 for binding (pM, Mean ±SD) Main parameter Anti-PD-L1 antibody EC₅₀ of competing with human PD-1715.56 ± 61.58

Example 4 Affinity of hAAG5D8 to a Variety of Other B7 Family Proteins

The affinity of hAAG5D8 to a variety of other B7 family proteins (PD-L1,PD-L2, B7-H3, PD-1 and CD80) was tested by ELISA. hAAG5D8 was diluted to100 μg/mL with a coating buffer (6 mM Na₂CO₃, 14 mM NaHCO₃), and thenadded 100 μL to each well followed by overnight incubation at 4° C. Theplate was then washed with PBST, and added with 250 uL of a blockingsolution (3% BSA/PBST) to each well to block for 2 h at 25° C.Afterwards, the plate was washed with PBST and added with 100 μL of aprotein sample (rhPD-L1, rhPD-L2, rhB7-H3, rhPD-1 or rhCD80) at aconcentration of 5 μg/mL to each well followed by incubation at 25° C.for 2 h. Next, the plate was washed with PBST and added with 100 μL ofan enzyme-labeled secondary antibody (the secondary antibodyanti-His-tag diluted 1:5000) to each well followed by incubation at 25°C. for 1 h. Finally, the plate was washed with PBST and then colordevelopment is performed. Values were read at 450 nm.

Results and conclusions: The binding of hAAG5D8 to a B7 family protein(PD-L1, PD-L2, B7-H3, PD-1 or CD80) is shown in FIG. 3. The results showthat hAAG5D8 can specifically bind to PD-L1 but not PD-L2, and hAAG5D8does not bind to other proteins of the same family with relatedfunctions (B7-H3, PD-1, and CD80). Accordingly, hAAG5D8 binds to PD-L1with extremely high specificity.

Example 5 Study on the Therapeutic Efficacy of hAAG5D8 on SubcutaneouslyTransplanted Tumor in Human Non-Small Cell Lung Cancer HCC827 NCG Mice

Twelve highly immunodeficient male NCG mice aged 4-5 weeks, weighing20-26 g (purchased from Model Animal Research Center of NanjingUniversity) were injected with 100 μl of human peripheral bloodmononuclear cells PBMC (1×10⁷ cells/100 μl, isolated from blood ofhealthy donors) via tail vein injection. Three days after tail veininjection, mice were inoculated subcutaneously with human non-small celllung cancer HCC827 cells (ATCC) on the right flank. Three days afterinoculation (an average tumor volume of 57 mm³), the mice were randomlydivided into 3 groups (A-1 group, A-2 group, and A-3 group) with 4 micein each group, and administered according to the dosage regimen shown inTable 9. Both positive control and negative control were set in thisexperiment, wherein the positive control was MPDL3820A (i.e.Atezolizumab) and the negative control was Human IgG1 (Crown Bioscience(Taicang) Co., Ltd., lot no. AB160083). On the day of the fourthadministration (that is, the 13th day after inoculation of tumor cells),the experimental mice were euthanized under CO₂ and then tumors weretaken. The relative tumor inhibition (TGI_(RTV)) and the tumor growthinhibition (TGI_(ΔTV)) were calculated for evaluation of drug efficacy.

The equation for calculating the relative tumor inhibition:TGI_(RTB)=1−T_(RTV)/C_(RTV) (%). (T_(RTV) is the relative tumor volume(TV) of the positive control group or treatment group at a specific timepoint, C_(RTV) is the relative tumor volume of the negative controlgroup at a specific time point, T_(RTV)/C_(RTV) is the percentage valueof the relative tumor volume of the positive control group or treatmentgroup and the relative tumor volume of the negative control group,RTV=V_(t)/V₀, V⁰ is the tumor volume of the animal at the time ofgrouping, and Vt is the tumor volume of the animal after treatment.)

The equation for calculating the tumor growth inhibition:TGI_(ΔTV)%=(1−ΔT/ΔC)×100%. (ΔT=the average tumor volume of the positivecontrol group or treatment group at a specific time point−the averagetumor volume of the positive control group or the treatment group at thebeginning of administration, and ΔC=the average tumor volume of thenegative control group at a specific time point−the average tumor volumeof the negative control group at the beginning of administration.)

Results and conclusions: The tumor inhibitory effects of hAAG5D8 onhuman non-small cell lung cancer models are shown in Table 10 and FIG.4, respectively. Table 10 shows the relative tumor inhibition and tumorgrowth inhibition of hAAG5D8 on human non-small cell lung cancer cells,and FIG. 4 shows tumor growth curves after administration. Experimentalresults show that hAAG5D8 has a significant inhibitory effect on humannon-small cell lung cancer.

TABLE 9 Dosage regimen for studying the tumor inhibitory activity ofhAAG5D8 on human non-small cell lung cancer Group Subject Drug Dose(mg/kg) Route Volume Period 1 A-1 group Human IgG1 5 Intravenousinjection 10 μL/g BIW × 4 times 2 A-2 group MPDL3820A 5 Intravenousinjection 10 μL/g BIW × 4 times 3 A-3 group hAAG5D8 5 Intravenousinjection 10 μL/g BIW × 4 times Note: Group 1 is the negative controlgroup, Group 2 is the positive control group, and Group 3 is thetreatment group.

TABLE 10 Relative tumor inhibition rand tumor growth inhibition ofhAAG5D8 on human non-small cell lung cancer tumor cells DrugTGI_(RTV)(%) TGI_(Δ) _(TV) (%) Human IgG1 (5 mg/kg), — — MPDL3820A (5mg/kg) 25.3 49.5 hAAG5D8 (5 mg/kg) 35.9 63.3

Example 6 Study on the Therapeutic Efficacy of hAAG5D8 on SubcutaneouslyTransplanted Tumor in Mouse Colorectal Cancer MC38 Transgenic Mice

Each of 30 human PD-1 transgenic female C57 mice (Animal ExperimentCenter of Tongji University) was implanted subcutaneously 3×10⁶ mousecolorectal cancer cells expressing human PD-L1 (Tongji University, code:MC38-hPD-L1) on the left flank. When the tumor volume reached about 100mm³, the mice were randomly divided into 5 groups: C-1 group (negativecontrol group, PBS, 6 mice), C-2 group (positive control group,Atezolizumab, 3 mg/kg, 6 mice), C-3 group (administration group,hAAG5D8, 1 mg/kg, 6 mice), C-4 group (administration group, hAAG5D8, 3mg/kg, 6 mice) and C-5 group (administration group, hAAG5D8, 9 mg/kg, 6mice). The mice were administered intraperitoneally 2 times a week, 5times in total. The tumor volume was measured twice a week until the16th day after the beginning of administration.

Calculation Equations

Tumor volume: TV=D1×D2²/2, wherein D1 and D2 represent tumor longdiameter and tumor short diameter, respectively.

Relative tumor volume: RTV=T_(VT)/T_(V1), where T_(V1) is the tumorvolume before administration, and T_(VT) is the tumor volume on eachmeasurement.

Relative tumor proliferation: T/C(%)=T_(RTV)/C_(RTV)×100%, whereinT_(RTV) represents the RTV of the treatment group or positive controlgroup, and C_(RTV) represents the RTV of the negative control group.

Relative tumor inhibition: TGI_(RTV)(%)=(1−T_(RTV)/C_(RTV))×100%.

Results and conclusions: The tumor inhibitory effects of hAAG5D8 onmouse colorectal cancer are shown in Table 11 and FIG. 5. The resultsshow that hAAG5D8 at a concentration of 3 mg/kg or above has asignificant tumor inhibitory effect on subcutaneously transplantedtumors of mouse colorectal cancer. At the dose of 3 mg/kg, the tumorinhibitory effect of the anti-PD-L1 antibody has been significantlysuperior to that of Atezolizumab.

TABLE 11 Tumor volume changes in tumor-bearing mice after administrationDose Tumor volume TV (mm³) T_(RTV)/ TGI_(RTV) Group (mg/kg) D0 D16C_(RTV)(%) (%) PBS — 99.9 ± 13.2 1974.2 ± 413.2  — — Atezolizumab 3 95.9± 16.8 1111.6 ± 435.2* 53 47 hAAG5D8 1 95.5 ± 9.8  1138.8 ± 144.9* 64 36hAAG5D8 3 90.7 ± 12.4  664.8 ± 92.0** 39 61 hAAG5D8 9 91.2 ± 15.9   319.1 ± 0190.4** 14 86 Note: The day of administration was defined asD0; *P < 0.05, **P < 0.01 vs PBS

REFERENCES

1. CHEN L, FLIES D B. Molecular mechanisms of T cell co-stimulation andco-inhibition [J]. Nature reviews Immunology, 2013, 13(4): 227-42.2. OHAEGBULAM K C, ASSAIL A, LAZAR-MOLNAR E, et al. Human cancerimmunotherapy with antibodies to the PD-1 and PD-L1 pathway [J]. Trendsin molecular medicine, 2015, 21(1): 24-33.3. ISHIDA M, IWAI Y, TANAKA Y, et al. Differential expression of PD-L1and PD-L2, ligands for an inhibitory receptor PD-1, in the cells oflymphohematopoietic tissues [J]. Immunology letters, 2002, 84(1): 57-62.4. TAUBE J M, ANDERS R A, YOUNG G D, et al. Colocalization ofinflammatory response with B7-h1 expression in human melanocytic lesionssupports an adaptive resistance mechanism of immune escape [J]. Sciencetranslational medicine, 2012, 4(127): 127ra37.

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6. BUTTE M J, KEIR M E, PHAMDUY T B, et al. Programmed death-1 ligand 1interacts specifically with the B7-1 costimulatory molecule to inhibit Tcell responses [J]. Immunity, 2007, 27(1): 111-22.7. CHENG X, VEVERKA V, RADHAKRISHNAN A, et al. Structure andinteractions of the human programmed cell death 1 receptor [J]. TheJournal of biological chemistry, 2013, 288(17): 11771-85.8. An Liu, Discovery on small molecular inhibitors of PD-1/PD-L1 pathway[D], Jilin: Jilin University School of Pharmaceutical Sciences, 2016,15.

1. An isolated anti-PD-L1 antibody or antigen-binding fragment thereofcomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region and/or the light chainvariable region comprise a CDR sequence identical to that of an antibodydefined by the following sequence or obtained by 1-2 amino acidsubstitutions of the CDR sequence of the antibody defined by thefollowing sequence: (1) an amino acid sequence of a heavy chain variableregion as shown in SEQ ID NO: 31; and/or (2) an amino acid sequence of alight chain variable region as shown in SEQ ID NO:32.
 2. The antibody orantigen-binding fragment thereof according to claim 1, wherein: (1) forthe heavy chain variable region, CDR1 comprises an amino acid sequenceas shown in SEQ ID NO: 1, 7, 13, 19 or 25 or obtained by 1 or 2 aminoacid substitutions of SEQ ID NO: 1, 7, 13, 19 or 25; CDR2 comprises anamino acid sequence as shown in SEQ ID NO: 2, 8, 14, 20 or 26 orobtained by 1 or 2 amino acid substitutions of SEQ ID NO: 2, 8, 14, 20or 26; CDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3,9, 15, 21 or 27 or obtained by 1 or 2 amino acid substitutions of SEQ IDNO: 3, 9, 15, 21 or 27; and/or (2) for the light chain variable region,CDR1 comprises an amino acid sequence as shown in SEQ ID NO: 4, 10, 16,22 or 28 or obtained by 1 or 2 amino acid substitutions of SEQ ID NO: 4,10, 16, 22 or 28; CDR2 comprises an amino acid sequence as shown in SEQID NO: 5, 11, 17, 23 or 29 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NO: 5, 11, 17, 23 or 29; CDR3 comprises an aminoacid sequence as shown in SEQ ID NO: 6, 12, 18, 24 or 30 or obtained by1 or 2 amino acid substitutions of SEQ ID NO: 6, 12, 18, 24 or
 30. 3.The antibody or antigen-binding fragment thereof according to claim 2,wherein: (1) CDRs 1-3 of the heavy chain variable region comprise aminoacid sequences of SEQ ID NOs: 1-3 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 1-3, and/or the CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 4-6 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 4-6; or (2)CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 7-9 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 7-9, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 10-12 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 10-12; or (3)CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 13-15 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 13-15, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 16-18 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 16-18; or (4)CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 19-21 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 19-21, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 22-24 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 22-24; or (5)CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 25-27 or obtained by 1 or 2 amino acidsubstitutions of SEQ ID NOs: 25-27, and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 28-30 orobtained by 1 or 2 amino acid substitutions of SEQ ID NOs: 28-30.
 4. Theantibody or antigen-binding fragment thereof according to claim 1,wherein CDRs 1-3 of the heavy chain variable region comprise amino acidsequences of SEQ ID NOs: 1-3; and/or CDRs 1-3 of the light chainvariable region comprise amino acid sequences of SEQ ID NOs: 4-6;preferably, (1) the heavy chain variable region comprises a sequence asshown in SEQ ID NO: 31, or comprising the same CDRs 1-3 as in SEQ ID NO:31 and more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toSEQ ID NO: 31; and/or (2) the light chain variable region comprises asequence as shown in SEQ ID NO: 32, or comprising the same CDRs 1-3 asin SEQ ID NO: 32 and more than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO:
 32. 5. (canceled)
 6. The antibody orantigen-binding fragment thereof according to claim 5, wherein: (1) theheavy chain variable region comprises an amino acid sequence as shown inSEQ ID NO: 31; and/or (2) the light chain variable region comprises anamino acid sequence as shown in SEQ ID NO:32; preferably, (1) the heavychain comprises an amino acid sequence as shown in SEQ ID NO: 33; and/or(2) the light chain comprises an amino acid sequence as shown in SEQ IDNO:
 34. 7. (canceled)
 8. The antibody or antigen-binding fragmentthereof according to claim 1, wherein the antibody is a monoclonalantibody, chimeric antibody, humanized antibody, bispecific antibody,multispecific antibody or Fab Fragment, F(ab′) fragment, F(ab′)₂fragment, Fv fragment, dAb, Fd, single chain antibody (scFv);preferably, the antibody is a humanized monoclonal antibody; preferably,the antibody or antigen-binding fragment thereof further comprising ahuman or murine constant region; preferably, the constant region isselected from IgG1, IgG2, IgG3, and IgG4; preferably, the constantregion is IgG1, IgG2A, or IgG2B. 9-12. (canceled)
 13. An isolatedpolynucleotide encoding the antibody or antigen-binding fragment thereofaccording to claim
 1. 14. A combination of an isolated polynucleotidecomprising a polynucleotide encoding the heavy chain of the antibody orantigen-binding fragment thereof according to claim
 1. 15. A nucleicacid construct comprising the polynucleotide according to claim 13,preferably, the nucleic acid construct is a vector.
 16. (canceled)
 17. Ahost cell comprising the nucleic acid construct of claim 15, preferably,the cell is a prokaryotic cell, eukaryotic cell, yeast cell, mammaliancell, E. coli cell or CHO cell, NS0 cell, Sp2/0 cell, BHK cell. 18.(canceled)
 19. An antibody-drug conjugate comprising the antibody orantigen-binding fragment thereof according to claim 1 and a drug toxin,preferably, the drug toxin is selected from chemicals, toxins,polypeptides, enzymes, isotopes, cytokines, antibodies, or otherbioactive substances or a mixture thereof that can inhibit cell growthor kill cells directly, indirectly, or by activating the body's immuneresponse to thereby treat tumors, preferably interleukins, tumornecrosis factors, chemokines, nanoparticles, MMAE, MMAF, DM1, DM4,calicheamicin, duocarmycin, and doxorubicin.
 20. (canceled)
 21. Apharmaceutical composition comprising the antibody or antigen-bindingfragment thereof according to claim 1, and a pharmaceutically acceptablecarrier.
 22. A method for the manufacture of an anti-PD-L1 antibody,comprising culturing the host cell according to claim 17 under acondition suitable for a vector encoding the anti-PD-L1 antibody orantigen-binding fragment to be expressed, and recovering the antibody orfragment.
 23. A method for enhancing T cell function, comprisingadministering an effective amount of the pharmaceutical compositionaccording to claim 21 to dysfunctional T cells.
 24. A method for thetreatment of T cell dysfunction, comprising administering atherapeutically effective amount of the pharmaceutical composition ofclaim 21 to a patient with T cell dysfunction, wherein the T celldysfunction comprises infection and tumor immunization.
 25. The methodaccording to claim 24, wherein the tumor immunization is caused by acancer selected from the group consisting of breast cancer, lung cancer,colon cancer, ovarian cancer, melanoma, bladder cancer, kidney cancer,liver cancer, salivary cancer, stomach cancer, glioma, thyroid cancer,thymic cancer, epithelial cancer, head and neck cancer, and gastric andpancreatic cancer.
 26. A method of treating or preventing a cancer,comprising administering a subject in need thereof the antibody orantigen-binding fragment thereof according to claim 1, preferably, thecancer is a solid tumor.
 27. (canceled)
 28. The method according toclaim 26, wherein the solid tumor is lung cancer, colorectal cancer,breast cancer, ovarian cancer, melanoma, bladder cancer, urothelialcancer, kidney cancer, liver cancer, salivary cancer, stomach cancer,gliomas, thyroid cancer, thymic cancer, epithelial cancer, head and neckcancer, gastric cancer, pancreatic cancer, Merkel cell carcinoma. 29.The method according to claim 28, wherein the lung cancer is non-smallcell lung cancer.
 30. The method according to claim 29, wherein theovarian cancer is triple negative breast cancer.